Enhanced wort fermentation with de novo lager hybrids adapted to high ethanol environments

AbstractInterspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2L-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance. Interestingly, it was not only the hybrid strains, but also the S. cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred both at chromosome and single nucleotide level in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation.ImportanceRecent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolate and characterize a number of adapted variants, which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variants strains were produced without any genetic modification, and are suitable for industrial lager beer fermentations.

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Enhanced Wort Fermentation with De Novo Lager Hybrids Adapted to High-Ethanol Environments

Applied and Environmental Microbiology ◽

10.1128/aem.02302-17 ◽

2017 ◽

Vol 84 (4) ◽

Cited By ~ 12

Author(s):

Kristoffer Krogerus ◽

Sami Holmström ◽

Brian Gibson

Keyword(s):

Adaptive Evolution ◽

Ethanol Tolerance ◽

De Novo ◽

Fermentation Performance ◽

Lager Yeast ◽

Brewing Yeast ◽

Ploidy Levels ◽

Ester Formation ◽

Lager Beer ◽

High Ethanol

ABSTRACT Interspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2-liter-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance than those derived from diploid strains. Interestingly, it was not only the hybrid strains, but also the Saccharomyces cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred at both the chromosome and single nucleotide levels in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation. IMPORTANCE Recent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here, we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolated and characterized a number of adapted variants which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variant strains were produced without any genetic modification and are suitable for industrial lager beer fermentations.

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Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

10.20944/preprints202012.0098.v1 ◽

2020 ◽

Author(s):

Martina Catallo ◽

Fabrizio Iattici ◽

Cinzia Randazzo ◽

Cinzia Caggia ◽

Kristoffer Krogerus ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Yeast Culture ◽

Culture Collections ◽

Lager Yeast ◽

Ethyl Hexanoate ◽

Brewing Yeast ◽

Saccharomyces Bayanus ◽

Lager Beer ◽

Saccharomyces Eubayanus

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomyces cerevisiae ale x Saccharomyces eubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus x Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae x S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20°C, one triple S. cerevisiae x S. eubayanus x S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2L wort fermentation trials at 12°C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for design of de novo lager beer hybrids and may help to diversify aroma profiles in lager beers.

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Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

Microorganisms ◽

10.3390/microorganisms9030514 ◽

2021 ◽

Vol 9 (3) ◽

pp. 514

Author(s):

Martina Catallo ◽

Fabrizio Iattici ◽

Cinzia L. Randazzo ◽

Cinzia Caggia ◽

Kristoffer Krogerus ◽

...

Keyword(s):

De Novo ◽

Yeast Culture ◽

Culture Collections ◽

Lager Yeast ◽

Ethyl Hexanoate ◽

Brewing Yeast ◽

Single Organism ◽

Saccharomyces Bayanus ◽

Lager Beer ◽

Beneficial Traits

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomycescerevisiae ale × Saccharomyceseubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus × Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae × S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20 °C, one triple S. cerevisiae × S. eubayanus × S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2 L wort fermentation trials at 12 °C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for designing de novo lager beer hybrids and may help to diversify aroma profiles in lager beer.

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Creation and Characterization of Industrially Applicable de Novo Lager Yeast Hybrids with a Unique Genomic Architecture

Applied and Environmental Microbiology ◽

10.1128/aem.02434-20 ◽

2020 ◽

Author(s):

Zachari Turgeon ◽

Thomas Sierocinski ◽

Cedric A. Brimacombe ◽

Yiqiong Jin ◽

Brittany Goldhawke ◽

...

Keyword(s):

De Novo ◽

Phenotypic Diversity ◽

Breeding Strategy ◽

Lager Yeast ◽

Genomic Architecture ◽

Group I ◽

Saccharomyces Pastorianus ◽

Lager Beer ◽

Group Ii ◽

Beer Production

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus. Lager strains are classified into two major groups based largely on genomic composition: Group I and Group II. Group I strains are allotriploid, whereas Group II are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off-flavours and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus. The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production. IMPORTANCE All lager beer is produced using two related lager yeast types: Group I and Group II, which are highly similar resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (Group III). We anticipate that these novel lager strains will be of great industrial relevance, and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

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Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid

Frontiers in Fungal Biology ◽

10.3389/ffunb.2021.733655 ◽

2021 ◽

Vol 2 ◽

Author(s):

Kristoffer Krogerus ◽

Frederico Magalhães ◽

Sandra Castillo ◽

Gopal Peddinti ◽

Virve Vidgren ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Phenotypic Diversity ◽

Single Cells ◽

Fermentation Performance ◽

Hybrid Strain ◽

Lager Yeast ◽

Brewing Yeast ◽

Acetate Ester ◽

Copy Numbers ◽

Saccharomyces Eubayanus

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

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Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid

10.1101/2021.07.01.450509 ◽

2021 ◽

Author(s):

Kristoffer Krogerus ◽

Frederico Magalhaes ◽

Sandra Castillo ◽

Gopal Peddinti ◽

Virve Vidgren ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Phenotypic Diversity ◽

Single Cells ◽

Fermentation Performance ◽

Hybrid Strain ◽

Lager Yeast ◽

Brewing Yeast ◽

Acetate Ester ◽

Copy Numbers ◽

Saccharomyces Eubayanus

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae x Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15 C, 15 Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

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Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2012.11.024 ◽

2013 ◽

Vol 161 (2) ◽

pp. 76-83 ◽

Cited By ~ 34

Author(s):

Hongjie Lei ◽

Liye Zheng ◽

Chenxia Wang ◽

Haifeng Zhao ◽

Mouming Zhao

Keyword(s):

Amino Acids ◽

Free Amino Acids ◽

Free Amino ◽

Fermentation Performance ◽

Lager Yeast

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What else is in an evolved name? Exploring evolvable specificity with SignalGP

10.7287/peerj.preprints.27122v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alexander Lalejini ◽

Charles Ofria

Keyword(s):

Adaptive Evolution ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

De Novo ◽

Direct Access ◽

Evolutionary Transitions ◽

Fine Grained ◽

Flexible Mechanism ◽

Gene Regulatory ◽

Program Modules

Tags are evolvable labels that provide genetic programs a flexible mechanism for specification. Tags are used to label and refer to programmatic elements, such as functions or jump targets. However, tags differ from traditional, more rigid methods for handling labeling because they allow for inexact references; that is, a referring tag need not exactly match its referent. Here, we explore how adjusting the threshold for how what qualifies as a match affects adaptive evolution. Further, we propose broadened applications of tags in the context of a genetic programming (GP) technique called SignalGP. SignalGP gives evolution direct access to the event-driven paradigm. Program modules in SignalGP are tagged and can be triggered by signals (with matching tags) from the environment, from other agents, or due to internal regulation. Specifically, we propose to extend this tag based system to: (1) provide more fine-grained control over module execution and regulation (e.g., promotion and repression) akin to natural gene regulatory networks, (2) employ a mosaic of GP representations within a single program, and (3) facilitate major evolutionary transitions in individuality (i.e., allow hierarchical program organization to evolve de novo).

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